Plant virus-based vectors allow for the rapid, transient expression of proteins in whole plants (Pogue et al. 2002; Scholthof et al. 2002). Although many different plant viruses have been modified to function as expression vectors, Tobacco Mosaic Virus (TMV) based vectors express the highest levels of foreign protein in plants (Pogue et al. 1998; Yusibov et al. 1999). TMV-based vectors were among the first viral vectors to be used for either gene expression or gene silencing in plants (Fitzmaurice et al. 2002; Kumagai et al. 1995; Kumagai et al. 1993). They have been effective vectors for the production of many different kinds of proteins in plants including allergens (Breiteneder et al. 2001; Krebitz et al. 2000), antibodies (Giritch et al. 2006) or antibody fragments (McCormick et al. 1999), and vaccine candidates (Gleba et al. 2005; Turpen et al. 1995).
TMV is an RNA virus that expresses large amounts of coat protein (CP), from a subgenomic promoter. To convert TMV to an efficient expression vector, an additional, heterologous coat protein subgenomic promoter and restriction enzyme sites for cloning of foreign DNA sequences were inserted into a T7 promoter driven cDNA clone of TMV (Shivprasad et al. 1999). In vitro transcription of this plasmid with T7 RNA polymerase is needed to generate biologically active transcripts. Transcripts are typically rub-inoculated by hand onto plants to initiate an infection (Pogue et al. 1998). The in vitro transcription and rub inoculation steps, in particular, add significantly to the cost and complexity of using TMV vectors.
Agroinfection (Grimsley 1995; Grimsley et al. 1986) is an alternative, less-expensive strategy for infecting plants with RNA viruses. In agroinfection, a plant functional promoter and RNA virus cDNA are transferred as T-DNA from Agrobacterium into plant cells. The T-DNA is transcribed in planta, to generate biologically active viral RNAs that can initiate self-replication. Although agroinfection has been used for many different plant RNA viruses, it has not been routinely used with TMV-based vectors.
Recently, an agroinfection-compatible TMV expression vector was constructed with extensive modifications to the TMV cDNA. These alterations included multiple mutations, to destroy cryptic introns, and insertion of multiple plant-gene introns into the TMV cDNA sequences in a binary vector (Marillonnet et al. 2005). These mutations improved the efficiency by which TMV vectors can be introduced into plants by agroinfection and are used in a process called “magnifection” (Gleba et al. 2005; Marillonnet et al. 2004). In magnifection, whole plants are submerged and infiltrated with Agrobacterium cultures carrying intron-modified TMV sequences in a binary vector. While the magnifection process is efficient, it is not easily adapted to a high throughput workflow. Also, the increased size of the intron-modified vectors can make cloning into these vectors more challenging. In addition, it is not clear if the intron-modified vectors are absolutely required for efficient local and systemic infection of plants with TMV when using standard agroinfection procedures.
In addition, as interest in proteomics, biochemistry and protein structure increases there is an increasing need for efficient, easy-to-use recombinant protein expression systems. Improving transient expression vectors so they are easier to use, more cost-effective and produce higher levels of recombinant proteins will be of great use.
Certain transient expression systems take advantage of the ability of Agrobacterium tumefaciens to transfer DNAs into plant cells. A. tumefaciens cell suspensions simply infiltrated (or injected) into leaves can efficiently transfer sequences from the T-DNA region of a modified A. tumefaciens Ti (binary) plasmid into plant cells. If the T-DNA transferred into the plant cell contains a DNA sequence of interest joined to a plant-functional promoter, the transferred DNA would be transcribed in the plant nucleus. One disadvantage of this approach, however, is that the expression of the T-DNA is generally quite low and transient and expression drops off after 5 days or so.
It was recently demonstrated that one reason for this was that post-transcriptional gene silencing (PTGS) directed toward the transcribed T-DNA was being induced in the plant after agroinfiltration. It was determined that this could be at least partially overcome by using two different A. tumefaciens cultures to simultaneously co-introduce T-DNAs for both a cauliflower mosaic virus 35S promoter (35S) driven gene of interest and a 35S driven RNA silencing suppressor gene into cells. Ectopic transient expression of an RNA silencing suppressor protein (such as the p19 protein from tomato bushy stunt virus) suppressed the PTGS of the introduced T-DNA. This resulted in an increase in the amount of recombinant protein expressed. For some proteins, ectopic co-expression of p19 resulted in a nearly 50-fold increase in recombinant protein expression levels (Voinnet et al., 2003).
Partially because of this improvement, this strategy has become one of the more commonly used plant transient expression systems. Using this strategy, hundreds of plant proteins have been expressed in a relatively high-throughput fashion (Popescu et al., 2007). One limitation of this strategy, however, is that relatively high concentrations of A. tumefaciens cell suspensions must be infiltrated into leaves in order to get the highest expression levels possible. For some plant species the infiltration of such high concentrations of A. tumefaciens can elicit negative (hypersensitive) responses from the plant (unpublished observations).
Other transient expression systems are based on plant viruses such as TMV, tobacco mosaic virus. TMV is a rod-shaped virus that has a single stranded ‘plus sense’ RNA genome. TMV expresses four proteins from three open reading frames. Two viral genes (the viral ‘movement protein’ and the capsid protein) are expressed from separate subgenomic promoters. To convert TMV into an expression vector, an additional subgenomic promoter was inserted into the viral genome to drive the expression of an inserted foreign gene. Plants can be inoculated with TMV vectors through a process called “agroinfection.” In agroinfection, A. tumefaciens was used to deliver a T-DNA comprised of a 35S promoter driven TMV cDNA to plant cells. Transcription of the T-DNA in the plant nucleus gave rise to an RNA that was capable of initiating self-replication in the cytoplasm. Multiple reports have documented the low agroinfection efficiency of the typical 35S-driven TMV vector (Turpen et al., 1993; Marillonnet et al., 2005; Man and Epel, 2006).
Therefore, in spite of intron-modified TMV vectors that have been recently constructed, there remains a need for TMV expression vectors with at least one or more of the following features: 1) contains convenient cloning sites for genes of interest; 2) can be used to infect plants in an easy and cost-effective manner; and, 3) leads to efficient systemic infection of inoculated plants.